non-canonicalautophagyincardiacmyocytesHaoYan*?,Jian-JunXu*,Wen-linLi?,Xiao-YuShi?,Yong-BingWu,Shu-QiangZhu*，XiangLong*andXiao-QiangZhang**Departmentofcardiacsurgery,TheSecondAffiliatedHospital,NanchangUniversity,Nanchang,jiangxi,330006,PRChina.?JiangxiProvincialKeyLaboratoryofMedicalbiologicalhightechnology,Nanchang,NanchangUniversity,Jiangxi,330006,PRChina.Correspondingauthors:Jian-JunXu: Departmentofcardiacsurgery,TheSecondAffiliatedHospital,nanchangUniversity,nanchang,jiangxi,330006,PRChina.: Fax: :Autophagy,DopamineReceptor,Raclopride,non-canonicalautophagyShorttitle:RacloprideInducedatypicalautophagyHeartdiseasestillistheleadingcauseofdeathininduatrializeworld[1],andadultcardiacmyocyteislong-livedandterminallydifferentiated，whichcannotbefullyreplacedbyitsproliferation，socatabolicpathwaysplayimportantroletomaintaincardiacmyocytecellularhomeostasis.Whilemostcellularproteindegradationismediatedthroughthreemajorpathways,includingcalpains,autophagyandubiquitin.Since1963deDuveintroducedthetermautophagy,autophagy(generallyreferredtomacroautophagy)befoundtodeliversthelong-livedproteinsanddamagedorganellestolysosomesfordegradation,andreplenishdepletedenergystoretomaintainingcellularhomeostasis,butexcesslevelsofautophagycanalsobetoxicandmayeveninducecelldeath,termedtypeIIprogrammedcelldeath.Cellkeepbasiclevelsautophagy,Autophagyalsocanbeinducedbycellstress,andmorethan30ATGgenescontroltheconservingdynamicprocessesofautophagy,however,recentlymanyreaserchreportatypicalautophagyoralternativemacroautophagyisindependentofULK1/2[2],LC3[3],l1[4]ormTOR[5].Thenon-canonicalautophagymechanismisstillmultiplicity,suchasbyCa2+-calpain-GsεpathwayorcAMP-Epac-PLC-ε-IP3pathway[5].DuringmanycardiacPathophysiologyofischemiaandreperfusion(I/R),hypertrophyandheartfailure,autophagyisexaminedtoinvolveinall.ButduringI/Rcardicautophagyeffectiscontroversial,duringischemiasomereaserchreportthatautophagyviaAMPKisbeneficial,butduringreperfusionautophagyviaJNKisdetrimental[6].sotheforms,levelorphaseofautophagymaydeterminewhetherautophagyisprotectiveordetrimentalinresponsetoheartDopamine(DA)isapotentcardiovascularneurotransmitter.Dopaminereceptorsplayanimportantroleincontrollingtheprincipalfunctionsofthecardiovascularcirculation,dopaminereceptorsweretraditionallydividedintotwofunctionalgroups,D1-like(D1andD5subtypes)andD2-like(D2,D3,andD4subtypes)families[6]D1-likeandD2-likereceptorshavebeenbothidentifiedinhumanandrathearts[7].D2-likereceptorsmayantagonizesD1-likereceptors,suchascoupledtotheadenylylcyclaseandL-typecalciumchannel.AlthoughDopamineinduceautophagyinneuroblastomacell[8]hasbeenreported，andDopamineReceptorAntagonistinvolveinindependentofmTORautophagy[9],theexactautophagymechanismsviaCa2+fluxordopaminereceptorsubtypesremaininconclisive,maybedependingondrugconcentration,timeofexposure,differentcelltypes.theaimofthepresentstudywastodeterminewhethertheD1--likeorD2-likeparticipateininduceautophagybydifferentmechanism.Inthisstudy,wehaveidenti?edanovelfunctionofracloprideasanautophagyinducerincardiacmyocytes.Wepresumedthatraclopride–inducedautophagyisnotincanonicalform.TheinvestigationconformstotheGuidefortheCareandUseofLaboratoryAnimals,publishedbytheNationalInstitutesofHealth(NIHPublicationNo.85-23,Revised1996).TheprocedureforcardiomyocyteisolationwasapprovedbytheSecondAffiliatedHospitalofUniversityofNanchang’sInstitutionalAnimalCareandUseCommittee.Primaryculturesofventricularcardiacmyocyteswerepreparedfrom1-2day-oldSprague-Dawleyrats(Animalsciencecenter,NanchangUniversity)byenzymaticdigestionwith0.25%trypsin(Invitrogen).NeonatalRatCardiomyocyteswerepreplatedfor2hrstoreducenon-myocytecontamination，thenwashingtoremovalerythrocytes，plated(2.0x106cells)incultureflasksandincubatedat37℃and5%CO2inhumidifiedatmosphere.CellswereculturedinDulbecco’smodifiedEagle’smedium(DMEM,Gibco,USA)with10%fetalbovineserum(FBS,SIGMA,USA)containingappropriateantibiotics，changedper24h.MostPrimaryculturesofventricularcardiacmyocytesbeatspontaneouslyinaconfluentmonolayer48–72hafterplating.Followingtreatment,NeonatalRatCardiomyocyteswerelysedinRIPAlysisbuffer(BeyotimeInstituteofBiotechnology,China).ProteinconcentrationsweredeterminedusingtheBCAproteinassayreagent(BeyotimeInstituteofBiotechnology,China).Equalproteinamounts(40-60μg)wereelectrophoresedinansodiumdodecylsulfatepolyacrylamidegel(8-15%)andtransferredtoapolyvinylidenefluoridemembrane,whichwasblockedwith2%albuminbovineⅤandincubatedwithrabbitanti-LC3antibody( ,USA)，Atg5，Atg7,P62(SIGMA,antibody(SANTACRUZ,USA)overnightat4oC.MembraneswerethenincubatedwithHRP-conjugatedanti-rabbitIgG(Sigma,USA)for1hatroomtemperature.Allprimaryantibodieswereusedatadilutionof1:500-1000andthesecondaryantibodywasusedatadilutionof1:5000.BlotsweredevelopedwithECLreagent(Thermo,USA)andexposedtofilm(Kodak,USA).Cardiacmyocyteswerecentrifugedforcollectionafterdrugincubated24h,fixedwith4%glutaraldehyde,post-fixedwith1%osmiumtetroxide,wereimmersedin0.1Mofsodiumcacodylatebuffer(pH7.3)containing2.5%glutaraldehydefor4h,andthenrinsedwiththecacodylatebufferalone,followedwithpost-?xationby1%osmiumtetroxideinthecacodylatebufferfor1h.Thecellwereembeddedineponresinandcutinto600-nm-thickslicesbyanultramicrotome.Thesliceswerestainedwithuranylacetateandleadcitrate,thenexaminedwithaelectronmicroscope(Hitachi,H7500,Japan)at75kV.Afterdrugsincubated24h,Cardiacmyocyteswerefixedwith4.0%paraformaldehydefor30minandacetonefor10min，and0.2%triton100forpermeation,thenblockedin2%albuminbovineⅤimmunostainingblockingbufferfor1hatroomtemperature.Forduallabelingstudies,thefixedcellswereincubatedwithrabbitanti-LC3antibody(1:50)( ,USA)andmouseanti-α-actinantibody( ,USA)overnightat4oCovernight.CellularproteinwasstainedbyincubatingwithFITCconjugatedanti-rabbitIgG(1:50)andTRITCconjugatedanti-mouseIgG(1:50)(Jackson,USA)for1hatroomtemperature.NucleiweredyedwithDAPI(Roche).Immunofluorescenceofcellswasvisualizedusingaconfocalmicroscopy(LeicaSP5,Mannheim,Freeintracellularcalciumconcentrations([Ca+2])inmyocardialcellswasdeterminedbyusingtheFluo-3/AM(sigma,USA)probe.Aftertreatedwithdrugs,NeonatalRatCardiomyocytewereincubatedwith5mol/LFluo-3/AMfor40minat37℃and5%CO2inhumidifiedatmosphere,washedthreetimeswithphosphate-bufferedsalineandfurtherincubatedfor20mininDMEM.allproceduresprotectfromlight.Dynamicchangesin[Ca+2]inmyocardialcellsweremeasuredafterstimulationwithdrugfor30minbymeasuringFluo-3fluorescence.Theimageswerecapturedusing40×oilimmersionobjective(488nmexcitation,522nmemission).TThechangeoffluorescentintensityinthecellscouldrepresentthechangeofintracellularCa2+concentration.Thefluorescenceintensitywasobservedineightrandomlychosencellsusingalaserscanningconfocalmicroscope(LeicaSP5,Mannheim,Germany)tocalculateaveragefluorescenceintensityforallcellsbyLASAFLite（LeicaSP5,Mannheim,Germany）.GeneSilencingwithsiRNA-rab7andsiRNA-ThesilencerselectsiRNAstargetingratrab9(Cat#: )andasilencerAccuTarget?Negativecontrol(Cat#:SN-1004)wereobtainedfromBioRP(Daejeon,SouthKorea).Fortransfection,cellswereplatedin35-mmtissueculturedishesat0.7x106cellsperdish,grownfor24h,thentransfectedwithsiRNAusingLipofectamineRNAiMAXandOpti-MEMinserum-freeandantibiotics-freemedium,Atg7siRNAandrab9siRNAatadoseof180pmolforperdish，accordingtothemanufacturer’sinstructions(Invitrogen).Threedaysaftertransfection,Atg7andRab9proteinsinthecellswerereducedtoalevelundetectablebyWesternblotysis(Fig.3A).indicatedbestrab9siRNAandAtg7siRNAinhibitautophagybyracloprideinduced.WeFoundtheDopamineDopaminedrugsinvolveinregulationofcardicPathophysiology,p62isanautophagysubstrate,andconversionofLC3-ⅠtoLC3-Ⅱisaimportantpartofautophagy，wetestedSKF-38393,Bromocriptine,SCH2390,racloprideandrapamycin(autophagyinducer0.1μM).neonatalratventricularmyocytesweretreatedwith40μMofeachDopamineDopamineReceptordrugsfor24hbeforeharvested.thelysateswereananlyzedbywesternblottingusinganti-LC3antbodytop62clearandanti-LC3antbodytodetecttheconversionofLC3-ⅠtoLC3-Ⅱ.Amongthe5drug,tworacloprideandrapamycinmoderayactivetop62clear,threeSKF-38393,BromocriptineandSCH2390wereshowednop62clear.similarlyintheconversionofLC3-ⅠtoLC3-Ⅱ,racloprideandrapamycintheconversionofLC3-ⅠtoLC3-Ⅱ,butSKF-38393,BromocriptineandSCH2390wereshowednotheconversionofLC3-ⅠtoLC3-ⅡweusedEMtoevaluatetheautophagyprocessesbyexaminingcharacteristicstructures.AsshowninFig.3,differenttypesofautophagosomes,whichcontainedheterogeneousorganellesrangingfrommitochondriatomultivesicularbodiessurroundedbyasequesteringmembrane,wereobservedinmyocytesunderraclopride(40μM)for24h(Fig.3c),rapamycin(0.1μM)for6h(Fig.3b)andcontrol(Fig.3a).BafilomycinA1wasoriginallyreportedtoinhibitautophagosome-lysosomefusionandlysosomalactivitybyraisinglysosomalpH,WeusedBafilomycinA1toinhibitlysosomalfunctiontomeasureautophagicfluxinneonatalratventricularmyocytescellculture,byimmunoblottingyses,asignificantincreaseinthenumberofLC3wasobservedinthecytosolafterthetreatmentwithraclopride,BafilomycinA1onlytraceactivitiesofLC3-Ⅱconversion,butBafilomycinA1reducedegradeofautolysosome,increaseLC3-Ⅱrecruit.Inthepresenceof200nMBafilomycinA1(2h),endogenousLC3-IIdramaticallyincreasedinracloprideinducedcells.TheamountofLC3-IIincellstreatedwithBafilomycinA1was0.8-foldhigherthaninuntreatedcontrolcellsat3h(Fig.4A).Theautophagyformationwasyzedbyanconfocalmicroscopyusinganti-LC3antibodyforLC3andanti-α-actinantibodytoidentificationforPrimarycardiacmyocytes，asignificantincreaseinthenumberofLC3wasobservedinthecytosolafterthetreatmentwithraclopride,recruitfromperipheraltoperinuclear,similarly,BafilomycinA1enchanceLC3plaqueaccumulation(Fig.4B).WeFoundtheDopamineDopamineReceptordrugsinvolveinregulationofcardicthatwefoundthatSKF-38393andbromocriptinestronglyincreased[Ca+2],SCH23390hadtenuityeffectinincreased[Ca+2].However,raclopridehadonlytraceactivitiesofdecreased[Ca+2](Fig.24E-BP1andp70S6KinasearedownstreamofmTOR,levelofPhospho-4E-BP1andPhospho-p70S6KinasearemTORactive,rapamycindown-regalationPhospho-4E-BP1andPhospho-p70S6Kinase,andinducedautophagydependentofmTOR,butDopamineDopamineReceptordrugsdoesnotchanglevelofPhospho-4E-BP1andPhospho-p70S6Kinase(Fig.1A).racloprideinducibleautophagyfluxwithoutdown-regalationPhospho-4E-BP1andPhospho-p70S6Kinase,itsautophagyisindependentofmTOR.racloprideenchanceautophagyInatime-dependentmanner(Fig.5).increasedconversionofLC3-ⅠtoLC3-Ⅱandp62clearfrom12h-24h，butATG5-ATG12didslightincrease.andrab9increaseduring0h-24h，butrab7increasein12h.Atg7IsRequiredforEfficientconventionalmacroautophagyFormationofAggresomes,Rab9IsRequiredforalternativemacroautophagy,butAtg7cannotchangeduring0h-24h，butrab9increasedat8h.weusesiRNAdown-reglateAtg7,0.4-foldlowerthanincontrolcells;wealsousesiRNArab9todown-reglaterab9,0.9-foldlowerthanincontrolcells,usedindicatedbestrab9siRNA(GUGCAUUUUAACCAACCAA)andAtg7siRNA(CAGACAAGAAGCUCCUUCU),wefoundthatsiRNAAtg7reduceracloprideconversionofLC3-ⅠtoLC3-Ⅱ,p62clearandincreasingrab9,butsiRNArab9didnotchangeracloprideconversionofLC3-ⅠtoLC3-Ⅱandp62clear(Fig.及教育水平有關(guān)[10]SNP[111-13],并與內(nèi)質(zhì)網(wǎng)應(yīng)激產(chǎn)生的自噬等胞內(nèi)轉(zhuǎn)運也相關(guān)，敲除D2多巴胺受體小鼠表現(xiàn)出帕金森病特征[14]，在大鼠模型中發(fā)現(xiàn)D2多巴胺受體參與代謝調(diào)節(jié)15]，使D2多巴胺受體阻斷劑raclopride通過增加血糖濃度來減少肌老化過程。多巴胺受體2激動劑bromocriptine可能參與了促進瓣膜病變[18,19]，還有對抗血自噬[10，被認為與受體對L-type鈣離子通道功能有關(guān)。我們研究發(fā)現(xiàn)對于心肌，明顯多于IP3Rs，發(fā)揮主導(dǎo)作用[21]。在缺血心肌病中“鈣超載”被認為激活各種信號，而在缺血再灌注中[22DR1CDRThapsigargin，ionomycin,ATP,vitaminD3[Ca2+]升高增加細胞自也認為[25]Thapsigargin被認為是升高鈣濃度引起內(nèi)質(zhì)網(wǎng)應(yīng)急，阻斷吞噬體與溶酶體結(jié)合L-typeCa2+通道，這也可能導(dǎo)致異常低鈣離子轉(zhuǎn)移到線粒體[5]。一般認為細胞內(nèi)Ca2+水平升高通過calpain依賴mTOR，包括無所不在的表達的calpain1和calpain2[28]。L-typeCa2+通道開發(fā)激活Calpain或過表達激活calpain2阻斷自噬和延遲清除體蛋白[5]，calpain激活剪切Atg5研究認為自噬并不完全依賴LC3-IconversiontoLC3-II[30]，這可能是其激活經(jīng)典自噬的機制之一。yuyanishida等，在通常經(jīng)典自噬之外還存在替代自噬途徑macroautophagyAtg7缺乏細胞。然而，這些過程仍可以被PI3kinase阻斷劑阻斷，同時依賴于Ulk1和運調(diào)控子Vps34的激活也可以不依賴于鈣離子[32]，其他方式的自噬，如Microautophagic于外界環(huán)境，脂[34]、等都影響自噬過程。多巴胺受體D2阻斷劑作為阻斷L-typeLloyd-Jones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